Arch Microbiol (1981) 129:135-140 Archives of Nia'nliulnOy ~ 9 Springer-Verlag 1981 Assimilatory Nitrate Uptake in Pseudomonas fluorescens Studied Using Nitrogen-13 Michael R. Betlach 1, James M. Tiedje 2, and Richard B. Firestone 3 1 Department of Microbiology and Public Health, z Departments of Microbiology and Public Health and Crop and Soil Sciences, and 3 Cyclotron Laboratory, Michigan State University, East Lansing, Michigan 48824, USA Abstract. The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, a3N as NO~, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate re- ductase, the form of t3N internally was ammonium and amino acids; the amino acid labeling pattern indicated that 13NO 3- was assimilated via glutamine synthetase and gluta- mate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO ~ at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in ceils grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar Km values, 7 gM. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium. Key words: Pseudomonasfluorescens - Assimilatory nitrate reduction - Nitrate reductase - Nitrate uptake - Active transport - Nitrogen-13 - Short-lived isotope Many genera of bacteria can assimilate nitrate (Hall 1978), but only a few have been examined to determine the mechanism of assimilation or its regulatory control (Brown et al. 1975; Guerrero et al. 1973; Sias and Ingraham 1979, van't Riet et al. 1968). In pseudomonads, growth on nitrate resembles growth under ammonium limitation: incorpo- ration of inorganic nitrogen into amino acids is catalyzed by glutamine synthetase and glutamate synthase, rather than by glutamate dehydrogenase (Brown et al. 1975). Growth at non-limiting ammonium concentrations represses nitrate Present addresses: 1 Extraterrestrial Research Div., NASA Ames Research Center, Moffett Field, California 94035, and 3 Lawrence Berkeley Laboratory, Berkeley, California 94720, USA Offprint requests to: J. M. Tiedje assimilation (Brown et al. 1975 ; van't Riet et at. 1968) as well as the glutamine synthetase-glutamate synthase pathway. More extensive investigations of nitrate assimilation in eucaryotes (for instance, Zumft 1976) indicate glutamine is the true repressor (Dunn-Coleman et al. 1979; Premakumar et al. 1979) and provide evidence for active transport of nitrate into the cells (Goldsmith et al. 1973; Schloemer and Garrett 1974). The mechanism of nitrate transport during assimilation by bacteria is not known. In dissimilatory nitrate reduction, chlorate, a nitrate analog, has little effect on nitrate reduction in intact bacteria but competitively inhibits the process in inside-out membrane vesicles (John 1977). Whether the nitrate carrier apparently responsible for chlorate exclusion in intact cells is part of an active transport system has not been determined. However, after examining pH transients in response to nitrate pulses in intact cells, Kristjansson et al. (1978) concluded that transport of the nitrate anion during denitrification occurred by facilitated diffusion through a proton symport. They believed the diffusion gradient was maintained by the rapid internal reduction of nitrate to nitrite, which then diffused out of the cells. Colorimetric procedures and the stable isotope ~SN have been used to monitor nitrate uptake, but such procedures lack the sensitivity to resolve rapid time-dependent events, such as inhibition of assimilation, or to monitor the movement of NO3-N into the organic N pool. Availability of a radioactive N tracer would alleviate many of the difficulties encountered with the use of lSN. The longest-lived radionuclide of nitrogen, Z3N has a half-life of only 10 min and so can be detected in amounts 10-*~ less than required for *SN mass spectrometry (Tiedje et al., Use of nitrogen-13 in studies of denitrification, Advances in Chemistry, Amer. Chem. Soc., in press). Recent development of procedures to use tan for studies of nitrogen fixation (Thomas et al. 1975) and denitrifi- cation (Tiedje et al. 1979) has provided access to this sensitive tracer to examine nitrogen assimilation. We report here on studies in which ~aNO~- was used to investigate the mech- anism of nitrate transport and assimilation in Pseudomonas J~IOFCSCgITS. Materials and Methods Organism and Growth Medium The strain of Pseudomonas fluorescens used (isolate 72) is representative of the dominant group of denitrifiers in soils and was isolated by Gamble et al. 1978, This strain has been submitted to the American Type Culture Collection. The growth medium contained 50 mM glucose; 50 mM 0302-8933/81/0129/0135/$01.00